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Title: Functional characterization of the teleost multiple tissue (tmt) opsin family and their role in light detection
Author: Fu, Josephine K. Y.
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2013
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In addition to a central circadian clock in the suprachiasmatic nucleus (SCN), zebrafish (Danio rerio) have local clock systems in their peripheral tissues. These peripheral tissues express a complement of clock genes that can be synchronized with the 24 h light/dark cycle and thus may be entrained by light. To date, teleost multiple tissue (tmt) opsin identified from Fugu rubripes and Danio rerio is the only opsin that has been proposed as a candidate to mediate this cellular photoentrainment (Moutsaki et al., 2003). Here we report the discovery of a multigene family of tmt opsins found not only in the teleost fishes, but in vertebrates,including amphibians, birds, reptiles, and some mammals. Phylogenetic analysis demonstrated that this gene family consists of three main classes, tmtI, tmtII and tmtIII, with each duplicating further to give two paralogues in the zebrafish genome. Their predicted amino acid sequences contain most of the characteristic features for the function of a photopigment opsin, as well as seven transmembrane segments indicative of a G protein coupled receptor (GPCR) superfamily. Significantly, reverse transcription polymerase chain reaction (RT-PCR) reveals that the tmt opsin genes in zebrafish are both temporally and spatially regulated. To investigate if these tmt photopigments mediate light-activated currents in cells, each opsin was expressed in vitro and the responses characterised by calcium imaging, whole-cell patch clamp electrophysiology, UV-Vis spectrophotometric analysis, and bioluminescence reporter assay. Collectively, these data suggest that some of the opsin photoproteins signal via Gi-type G protein pathway. Interestingly, the spectral analysis obtained shows that most tmt opsins tested are UV-sensitive when reconstituted in vitro with 11-cis and all-trans retinal, indicating an intrinsic bistable dynamics. Using site directed mutagenesis on one of the tmt opsins, tmt10, the potential spectral tuning sites involved in UV detection were tested. As part of this study, tmt opsin cDNAs were isolated from three populations of Mexican tetra (Astyanax mexicanus): surface, Pachon and Steinhardt. This allowed for a direct comparison between the tmt opsins present in the dark adapted species (cavefish) versus those of the light adapted species (zebrafish). It is hoped that the findings from this project will contribute to our understanding of non-visual light detection in fish and the evolution of their non-image forming photoreception.
Supervisor: Hankins, Mark W. Sponsor: Biotechnology and Biological Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Life Sciences ; Biochemistry ; Bioinformatics (biochemistry) ; Molecular biophysics (biochemistry) ; Bioinformatics (life sciences) ; Biology ; Cell Biology ; Genetics (life sciences) ; Medical sciences ; Biology (medical sciences) ; DNA damage signalling ; Genetics (medical sciences) ; Ophthamology ; Physiology ; Physical Sciences ; Biophysics ; Membrane proteins ; Molecular genetics ; Photochemistry and reaction dynamics ; Protein chemistry ; Protein folding ; Spectroscopy and molecular structure ; Opsin ; G protein-coupled receptor ; Photopigment ; Multiple Tissue expression ; Transduction ; Photosensory ; Biological rhythms